Cell sorting device and method of manufacturing the same

ABSTRACT

A system, method and apparatus employing the laminar nature of fluid flows in microfluidic flow devices in separating, sorting or filtering colloidal and/or cellular particles from a suspension in a microfluidic flow device is disclosed. The microfluidic flow device provides for separating a particle within a suspension flow in a microfluidic flow chamber. The chamber includes a microfluidic channel comprising at least one inlet port for receiving a suspension flow under laminar conditions, a first outlet port and a second outlet port. The chamber further includes an interface for translating a particle within the channel. The first outlet port receives a first portion of the suspension exiting the said channel and the second outlet port receives the particle in a second portion of the suspension exiting the channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Pat. application Ser. No.10/248,653, filed Feb. 4, 2003, now U.S. Pat. No. 7,318,902, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/354,372, filed on Feb. 4, 2002, both applications being incorporatedby reference in their entirety herein.

FIELD OF THE INVENTION

The present invention relates to a general class of devices thatuniquely employ laminar flows in separating, filtering or sortingcolloidal or cellular particles from a suspension within microfluidicdevices.

BACKGROUND OF THE INVENTION

Microfluidic flows are particularly useful due to their ultra laminarnature that allows for highly precise spatial control over fluids, andprovides both unique transport properties and the capability forparallelization and high throughput. These qualities have mademicrofluidic platforms a successful option for applications in printing,surface patterning, genetic analysis, molecular separations and sensors.Specifically, the effective separation and manipulation of colloidal andcellular suspensions on the microscale has been pursued with keeninterest due to the tremendous multidisciplinary potential associatedwith the ability to study the behavior of individual particles andcells. Devices that employ electric fields to direct flow for thepurpose of sorting and manipulating populations of cells have beenrealized and in some cases have demonstrated potential to achieveefficiencies comparable to their conventional analog, fluorescentactivated cell sorters (FACS).

SUMMARY OF THE INVENTION

The present invention relates to a system, method and apparatusemploying the laminar nature of fluid flows in microfluidic flow devicesin separating, sorting or filtering colloidal and/or cellular particlesfrom a suspension in a microfluidic flow device. In one embodiment, amicrofluidic flow device is provided for separating a particle within asuspension flow in a microfluidic flow chamber. The chamber includes amicrofluidic channel comprising an inlet port for receiving a suspensionflow under laminar conditions, a first outlet port and a second outletport. The chamber further includes an interface for translating aparticle within the channel. The first outlet port receives a firstportion of the suspension exiting the channel and the second outlet portreceives the particle in a second portion of the suspension exiting thechannel.

An alternative microfluidic flow device for separating a particle from asuspension flow into a second fluid flow is also provided. Themicrofluidic flow device includes a microfluidic channel comprising afirst inlet port for receiving the suspension flow, a second inlet portfor receiving the second fluid flow, a first outlet port and a secondoutlet port. The channel is adapted to receive the suspension flow andthe second fluid flow under laminar conditions. The device furtherincludes an interface for translating a particle from the suspensionflow to the second fluid flow. The first outlet port is adapted toreceive at least a portion of the suspension flow exiting the channeland the second outlet port is adapted to receive the particle in atleast a portion of the second fluid flow exiting channel.

A method of separating a particle within a suspension is also providedin which a suspension flow is received in a microfluidic channel underlaminar conditions. A particle in the suspension is translated withinthe suspension flow. A first portion of the suspension flow exitsthrough a first outlet port, and the particle exits in a second portionof the suspension flow through a second outlet port.

Another method of separating a particle from a suspension flow isprovided in which a suspension flow and a second fluid flow are receivedin a microfluidic channel. The suspension and the second fluid flowunder laminar conditions in the channel. A particle is separated fromthe suspension flow into the second fluid flow. At least a portion ofthe suspension flow exits through a first outlet port, and the particleexits in at least a portion of the second fluid flow through a secondoutlet port.

A cartridge is also provided for use in system to separate a particlefrom a suspension flow. The cartridge comprises a microfluidic channelincluding an inlet port for receiving a suspension flow under laminarconditions, a first outlet port and a second outlet port. The cartridgefurther comprises an interconnect for connecting the cartridge to thesystem. The microfluidic channel is adapted to receive the suspensionflow and provide an environment for translating the particle within thesuspension flow. The first outlet port is adapted to receive a firstportion of the suspension flow, and the second outlet port is adapted toreceive the particle in a second portion of the suspension flow.

An alternative cartridge is further provided for use in system toseparate a particle from a suspension flow into a second fluid flow. Thecartridge comprises a microfluidic channel including a first inlet portfor receiving the suspension flow, a second inlet port for receiving thesecond fluid flow, a first outlet port and a second outlet port. Thechannel is further adapted to receive the suspension flow and the secondfluid flow in the channel under laminar conditions. The cartridgefurther comprises an interconnect for connecting the cartridge to thesystem. The microfluidic channel is adapted to provide an environmentfor translating the particle from the suspension flow to the secondfluid flow. The first outlet port is adapted to receive at least aportion of the suspension flow, and the second outlet port is adapted toreceive the particle in at least a portion of the second fluid flow.

A system for separating a particle from a solution in a microfluidicflow device is also provided. The system includes a detector, aninformation processor and an actuator. The detector monitors amicrofluidic channel of the microfluidic flow device and provides anoutput to the information processor. The information processor processesthe output to determine if the particle is present. If the particle ispresent, the information processor triggers the actuator to translatethe particle within the channel.

A microfluidic chemical dispenser for dispensing a fluid flow into aplurality of receptacles is further provided. The dispenser comprises afirst inlet port, a second inlet port, a third inlet port, a centralchannel, a plurality of outlet ports, and a modulator. The channel isadapted to receive, under laminar conditions, a first fluid flow throughthe first input port, a second fluid flow through the second input portand a third fluid flow through the third input port. The second inputport is positioned at a first angle to the first input port, and thethird input port is positioned at a second angle to the first inputport. The modulator modulates the flow rates of the second and thirdfluid flows to dispense the first fluid flow into a plurality of outletports.

The foregoing and other features, utilities and advantages of theinvention will be apparent from the following more particulardescription of a preferred embodiment of the invention as illustrated inthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flow diagram of an actuated process of separating acolloidal or cellular particle from a suspension in a microfluidic flowdevice;

FIG. 2 depicts a block diagram of an exemplary system for separating acolloidal or cellular particle from a suspension in a microfluidic flowdevice;

FIG. 2 a depicts a block diagram of a microfluidic flow network that maybe used in conjunction with the system depicted in FIGS. 2, 3 and 4;

FIG. 3 depicts a block diagram of an alternative system for separating acolloidal or cellular particle from a suspension in a microfluidic flowdevice;

FIG. 4 depicts a block diagram of another alternative system forseparating a colloidal or cellular particle from a suspension in amicrofluidic flow device, wherein the system controls a valve actuatorto separate the particle from the suspension;

FIG. 5 depicts a fluid flow path in one example of a microfluidic flowchamber;

FIG. 5 a depicts a particle entering the microfluidic flow chamberdepicted in FIG. 5 via an inlet port;

FIG. 5 b depicts the particle depicted in FIG. 5 a being moved within acentral channel of the microfluidic flow chamber depicted in FIG. 5;

FIG. 5 c depicts the particle depicted in FIG. 5 a exiting the centralchannel of the microfluidic flow chamber depicted in FIG. 5 via anoutlet port;

FIG. 6 depicts side-by-side laminar fluid flows in the central channelof the microfluidic flow chamber depicted in FIG. 5;

FIG. 6 a depicts a particle entering the central channel via an inletport of the microfluidic flow chamber in the first fluid flow depictedin FIG. 6;

FIG. 6 b depicts the particle depicted in FIG. 6 a being moved withinthe central channel of the microfluidic flow chamber from the first flowto the second flow;

FIG. 6 c depicts the particle depicted in FIG. 6 a exiting the centralchannel of the microfluidic flow chamber in the second flow via anoutlet port;

FIG. 7 depicts an alternative example of a microfluidic flow chamber;

FIG. 7 a depicts side flows pinching a central flow of a suspension atthe entrance to a central channel of the microfluidic flow chamberdepicted in FIG. 7 to orient the flow of suspension in the centerportion of the channel;

FIG. 7 b depicts side flows pinching a central flow of a suspension atthe entrance to a central channel of the microfluidic flow chamberdepicted in FIG. 7 to orient the flow of suspension in the bottomportion of the channel;

FIG. 7 c depicts side flows pinching a central flow of a suspension atthe entrance to a central channel of the microfluidic flow chamberdepicted in FIG. 7 to orient the flow of suspension in the top portionof the channel;

FIG. 8 depicts another example of a microfluidic flow chamber includinga plurality of outlet ports for sorting colloidal and/or cellularparticles in a suspension;

FIG. 9 depicts a microfluidic flow chamber including a mechanicalactuator for separating a colloidal and/or cellular particle in asuspension, wherein the mechanical actuator comprises a valve;

FIG. 9 a depicts an alternative example of a microfluidic flow chamberincluding a mechanical actuator for separating a colloidal and/orcellular particle in a suspension, wherein the mechanical actuatorcomprises a valve;

FIG. 9 b depicts the particle being separated from the suspension viathe valve of the microfluidic chamber depicted in FIG. 9 a being closedto divert the particle into an alternative outlet port;

FIG. 9 c depicts the particle exiting the alternative outlet port of themicrofluidic chamber depicted in FIG. 9 a and the valve retracting toits open position;

FIG. 9 d depicts another alternative example of a microfluidic flowchamber including a chemical actuator for separating a colloidal and/orcellular particle in a suspension, wherein the chemical actuatorcomprises a chemically actuated valve;

FIG. 9 e depicts the particle being separated from the suspension viathe valve of the microfluidic chamber depicted in FIG. 9 d being swollenclosed to divert the particle into an alternative outlet port;

FIG. 9 f depicts the particle exiting the alternative outlet port of themicrofluidic chamber depicted in FIG. 9 d and the valve shrinking to itsopen position;

FIG. 10 depicts a series of suspensions being introduced into amicrofluidic flow chamber in series separated by buffers;

FIG. 11 depicts an alternative non-actuated microfluidic flow device forseparating colloidal and/or cellular particles from a suspension;

FIG. 12 depicts another alternative non-actuated microfluidic flowdevice for separating colloidal and/or cellular particles from asuspension;

FIG. 13 depicts an exemplary non-actuated microfluidic flow device forsorting colloidal and/or cellular particles from a suspension by size;

FIG. 14 depicts an alternative non-actuated microfluidic flow device forseparating motile cellular particles from a suspension;

FIG. 15 depicts an exemplary non-actuated microfluidic flow device forseparating colloidal and/or cellular particles from a suspension; and

FIG. 16 depicts a cartridge including a microfluidic flow chamber.

DETAILED DESCRIPTION

The processes and devices described herein relate to actuated ornon-actuated separation of various colloidal and/or cellular particlesfrom a suspension flowing under laminar conditions in a microfluidicflow device. The colloidal and cellular particles may include, forexample, polymeric, inorganic or other abiotic colloidal particles,individual polymers, proteins, fragments of DNA or RNA, entire sectionsor genomes of DNA, cells including single-celled organisms, formedbodies such as they would appear in blood, viruses and the like. Amicrofluidic flow device, as used for the purposes of the presentinvention, refers to a microscale device that handles volumes of liquidon the order of nanoliters or picoliters.

Under “laminar” flow conditions, a fluid flows through a channel withoutturbulence. The quantification of laminar or nonturbulent behavior istypically done through calculation of the Reynolds number, Re=ρνD/η,where ρ is the fluid density, η is the fluid viscosity, ν is the fluidvelocity, and D is some characteristic channel dimension (typically thechannel width). If the Reynolds number is small (<1000) for typicalchannel geometries, then flow is laminar, reversible, and non-turbulent.For this reason, the diameter of the channel can be designed to accountfor the intended fluid properties and fluid velocity, or, equivalently,the fluid velocity can be determined by the fluid properties and thechannel diameter.

FIG. 1 shows a flow diagram of a process for an actuated separation ofcolloidal and/or cellular particles from a suspension flowing through amicrofluidic flow device under laminar conditions. In the receive inputblock 10, an input is received from a sensor monitoring a target regionfor a particle of interest. The target region may be monitored to detectany known attribute (or absence thereof) that can be used to distinguisha particle from the remaining suspension. An imaging device such as acharge-coupled device (CCD) camera, for example, may be utilized tocapture a stream of images that may be used to identify a particle byits particular morphological attributes or motility. Alternatively,signatures, fingerprints or indices such as a fluorescent signature,light scattering signature, optical fingerprint, X-ray diffractionsignature or index of refraction, and the like, or any combination ofthese, may be used to distinguish the particle from the remainingsuspension. Surface charges of particles may also be used to distinguishthe particle by observing the reaction of the particle to an appliedelectric or magnetic field.

Further, the suspension or the individual particles may be pretreated,as known in the art, to enhance the recognition of the particles. Thesuspension may further be pretreated with an antibody that will bindspecifically to a particular type of particle may be used to enable orenhance the recognition of the particle. A suspension of cells, forexample, may be pretreated with antibody-decorated magnetic particlesand passed through a magnetic field to distinguish the particles fromthe remaining suspension. Similarly, other recognition methodologiesknown in the art may be used to distinguish the particle of interestfrom the remaining suspension.

Information processing block 20 performs any processing steps necessaryto distinguish the particle from the remaining suspension such ascomparing received images or signals from the receive input block 10 tothreshold values, e.g., size and shape. The information processing block20 may include any required processing steps as known in the art todistinguish the particle of interest from the remaining suspension. Theprocessing steps may vary depending upon the type of input received. Theprocessing step, for example, may include simple recognition of adigital input value or may include complicated processing steps todetect whether a given input corresponds to the presence of a particleof interest.

After a particle is identified, the particle may be separated from thesuspension by the actuation of separation block 30. The actuation mayinclude, for example, steering an optical trap such as via apiezoelectric mirror, an acoustic optic deflector, a diffractiongrating, a holographically-generated trap, a static line trap, a dynamicline trap, an optical gradient, a microlens array, a waveguidingstructure or other known optical steering mechanism. The actuation mayalternatively include generating an electric field or a magnetic field.The actuation may also include a mechanical or chemical actuator. Amechanical actuator, for example, may include a pump, valve, gate,applied pressure and the like. A chemical actuator, for example, mayinclude a hydrogel or similarly behaving material that reacts to aproperty sensed in the suspension that may indicate the presence orabsence of a particle of interest.

Each of the functions shown in blocks 10, 20 and 30 of FIG. 1, however,need not be performed by distinct hardware components. A sensor, forexample, may receive an input and perform the information processing onthat input to determine if a particle of interest has been detected. Anactuator may even perform each of the functions by directly reacting toa property being monitored (e.g., a pH responsive hydrogel may swell inresponse to a sensed pH level).

FIG. 2 shows one exemplary system 40 for separating a particle ofinterest from a suspension in a microfluidic flow device 44 utilizing anactuated separation technique. The system includes an detector system50, an information processing system 60 and an actuator system 70. Thedetector system 50 includes an imaging system, such as a camera 52, thatmay be used to image a field of view through a filter 54 and amicroscope 56. The detector system 50, for example, may utilize a CCDcamera to capture a stream of images of the microfluidic flow devicethrough a microscope lens. In one particular embodiment, the camera 52captures images at a rate of 30 images per second through a100×objective. The images are recorded by a recording device, such asVCR 58, and/or passed directly to an information processor, such as acomputer 62. Optionally, the identification of the particles may beaided by utilizing the laser 74 or another light source, such as asecondary laser, multiple other lasers, a broad spectrum lamp and thelike, to irradiate the suspension to illuminate the particles ofinterest.

The information processor may include the computer 62, a controller orother processor known in the art. The information processor receives andprocesses the image data and distinguishes the particle of interest fromthe remaining suspension as described above. Once the particle isrecognized, the information processor may trigger the actuator system 70to separate the particle from the suspension.

The actuator system 70 may include a targeting device 72 to target alaser beam from a laser 74 on the microfluidic flow device 44. Thetargeting device, for example, may include a piezo drive 76 to control apiezo mirror 78 to direct the beam of a laser 74. The laser 74, whenfocused on the particle, traps the particle. The optical trap may thenbe used to translate the particle between streams in the channel of themicrofluidic flow device 44.

Utilizing an optical trap as the means of actuation provides thecapability for highly precise and immediately customizable individualseparations Other applied fields, however, may also be utilized totranslate particles from the primary stream to the secondary stream.Both electric and magnetic fields may be employed with appropriatesuspensions to isolate individual or multiple particles. All colloidalparticles and living cells carry with them a surface charge, which, inthe presence of an electrical field results in electrophoresis. Theelectrophoretic force, or the migration of surface ions with an electricfield, is sufficient to translate cells or particles from one stream toanother. Similarly, if a particle or cell possesses a magnetic moment,it may be selectively translated in a magnetic field. Each of thesefields could be applied continuously to fractionate particles or cellsbased on electrical or magnetic properties, or could be pulsed orapplied discriminatively for custom separations.

As described above, the suspension or the individual particles may bepretreated, as known in the art. The pretreatment, for example, mayenhance the response of the particle to an optical trap or electric ormagnetic field. The suspension may further be pretreated with items,such as antibodies that will bind specifically to a particular type ofparticle may be used to enable or enhance the movement of the particlevia an optical trap or electric or magnetic field. A suspension ofcells, for example, may be pretreated with antibody-decorated magneticparticles and, thus, be easily moved by means of a magnetic field.

FIG. 2 a shows further detail of a microfluidic flow device 44 that maybe used in connection with a system 40, 80 and 110 such as shown inFIGS. 2, 3 and 4, respectively. The microfluidic flow device 44 includesa flow generator 45, which provides a pressure differential to inducefluid flows through the microfluidic flow device 44. The pressuredifferential, for example, may be induced by any method known in the artsuch as, but not limited to, capillary forces; gravity feed;electro-osmosis systems; syringes; pumps such as syringe pumps (e.g., akdScientific, model 200 syringe pump), peristaltic pumps and micropumps;valves such as three-way valves, two-way valves, ball valves andmicrovalves; suction; vacuums and the like. Further, although FIG. 2 ashows the flow generator located upstream of a microfluidic flow chamber47, the flow generator may also be placed midstream in the microfluidicflow chamber 47 or downstream of the microfluidic flow chamber 47.Further, the microfluidic flow chamber 47 preferably provides at leastone output 49 with the collected particles separated from a suspensionwithin the chamber. This output 49 may provide the collected particlesas an end process or may provide the particles to a downstream networkfor further processing.

FIG. 3 shows an alternative system for separating a particle of interestfrom a suspension in a microfluidic flow device. The imaging system 90and its operation is the same as shown in FIG. 2 except that the imagingsystem 90 further includes a field generator 92. The field generator 92induces an electric or magnetic field in the microfluidic flow device44. As the suspension flows through the device 44, the movement of theparticles of interest, whether induced by electric or magneticproperties of the particles themselves or by properties associated witha pretreatment of the particles, is captured by the imaging system 90and identified by the information processor 100.

FIG. 4 shows another system 10 for separating a particle of interestfrom a suspension in a microfluidic flow device 114. In this system, theactuator system includes a valve controller 112 that controls theoperation of a valve within the microfluidic flow device 114. The valve,for example, may be opened to divert the flow of the suspension withinthe microfluidic flow device for a predetermined time after therecognition of the particle of interest. In this manner, the systemseparates the particle in a small portion of the suspension by divertingthe suspension carrying the particle into an alternative outlet port. Anexample of such a valve is described below with respect to FIGS. 9 a-9c.

A particular microfluidic flow channel can be modeled to determine theflow path of a fluid flowing in a laminar manner through the channel.This is well known in the art and involves solving the Langevinequations, the Navier-Stokes equations or other equations of motion,which can be done manually or electronically. Commercial software toolsare also available for modeling the laminar flow path of a fluid throughany microfluidic flow channel. For example, CFDASE, a finite elementmodeling for computational fluid dynamics module available from OpenChannel Foundation Publishing Software from Academic & ResearchInstitutions of Chicago, Ill., and FIDAP, a flow-modeling tool availablefrom Fluent, Inc. of Lebanon, N.H., can be used to model the laminarflow of a fluid through a particular microfluidic channel.

FIG. 5 shows an embodiment of a microfluidic flow chamber 120 in which aparticle of interest may be separated from a suspension. Themicrofluidic flow chamber includes a single inlet port 122, two outletports 124 and 126 and a central channel 128. FIG. 5 further shows arrowsdepicting a modeled laminar flow of a particular fluid through themicrofluidic flow chamber 120. FIGS. 5 a-5 c show a process forseparating a particle 130 from a suspension flow in the microfluidicflow chamber 120 of FIG. 5. FIG. 5 a shows the particle entering themicrofluidic flow chamber 120 via the inlet port 122 at which point itis identified as described above. The information processor initiates anactuator to direct the particle 130 into a desired portion of the flowstream 132 of the suspension in FIG. 5 b. Thus, the particle 130 isdirected to a portion of the flow in which it will exit the centralchamber 128 through the second outlet port 126, as shown in FIG. 5 c.

FIGS. 6 and 6 a-6 c show an alternative embodiment of a microfluidicflow chamber 140, which includes two inlet ports 142 and 144, a centralchannel 146 and two outlet ports 148 and 150. As FIG. 6 shows, a firstfluid 152, indicated by dye, enters the central channel 146 via thefirst inlet port 142 and a second fluid 154 enters the central channel146 via the second inlet port 144. As described above, when the firstfluid 152 and the second fluid 154 flow through the microfluidic flowchamber in a laminar manner, the fluids maintain separate streams andundergo minimal convective mixing. Rather, the mixing present isprimarily due to molecular-scale diffusion, which for colloidal-sizedparticles is referred to as Brownian movement, as shown near the outletport of the central channel. The system can be designed to minimize thediffusion that occurs within the central channel 146 by controlling thecentral channel 146 dimensions and the velocity of the fluid flowingthrough the channel 146. In general, the diffusion distance x, can beexpressed as x ≈√{square root over ( )}D•t, wherein D is the diffusivityand t is the time. To a first order, the diffusivity is inverselyproportional to the size of the particle. Therefore, to a first order,the channel residence time required to achieve complete mixing, t≈x²D⁻¹,scales linearly with the particle diameter. Thus, by designing themicrofluidic flow chamber dimensions for a particular flow rate of afluid, a laminar two-phase flow may be used as an effective barrieragainst particle cross-transport. In the example shown in FIG. 6, eachof the inlet streams has a width of about 30 μm and the central channelhas a length from the inlet ports to the outlet ports of about 3000 μm,the reduction of which will correspondingly reduce the diffusion withinthe channel 146 for a constant flow rate. Both of the fluids streams 152and 154 shown are water. The first stream 152 includes a molecular dye(Methylene Blue), which has a diffusion coefficient on the order ofabout 1×10⁻⁵cm²/sec in water.

Further, as shown by the dashed line in FIG. 6 a, a portion of thesecond fluid stream 154 can exit the central channel 146 via the firstoutlet port 148 while the remainder of the second fluid 154 exits viathe second outlet port 150. If the first fluid 152 is a suspensionincluding suspended particles and the second fluid 154 is a cleansolvent, for example, the portion of the solvent that exits the firstoutlet port 148 along with the suspension 152 acts as an additionalbarrier to cross-contamination of the streams through diffusion. Thus,particles that diffuse into this portion of the solvent stream may stillexit the central chamber 146 via the first outlet port 148, as shown inFIG. 6.

The steady state flow-based particle barrier can be penetrated, however,by providing an actuator to move a particle 156 across the barrier. Aselective activation of an electric, magnetic or optical field, or anycombination of these fields, for example, may be used to move theparticle 156 from one stream to another stream. Alternatively, amechanical actuator, such as a valve, pump, gate or applied pressure maybe employed to move the particle from one stream to another stream.Although described here for parallel flows, the flows traveling inarbitrary orientations, including opposite directions, are possible.

FIGS. 6 a-6 c show a particle 156 being separated from the first inletstream 152 into the second inlet stream 154 in the embodiment shown inFIG. 6. In FIG. 6 a, a suspension enters the central channel 146 fromthe first inlet port 142, and a second fluid 154, such as a solvent,enters the central channel 146 from the second inlet channel 144. Thesuspension 152 and the second fluid 154 flow in a laminar manner throughthe central channel 146. The suspension stream 152 and a portion of thesecond fluid stream 154 exit the central channel 146 via the firstoutlet port 148. The remaining portion of the second fluid stream 154functions as a collection stream and exits the central channel 146 viathe second outlet port 150. A particle 156 suspended in the suspensionstream 152 is shown entering the central channel 146 from the firstinlet port 142, where it is identified as described above. In FIG. 6 b,the particle 156 is shown being separated from the suspension stream 152into the second fluid stream 154. The particle 156 may be separated fromthe suspension 152 via an electrical, magnetic, mechanical or chemicalactuator such as described above. In FIG. 6 c, the particle 156 is shownexiting the central channel 146 via the second outlet port 150 in thesecond fluid stream 154 for collection.

FIG. 7 shows another embodiment of a microfluidic flow chamber 160 inwhich a particle of interest may be separated from a suspension. Themicrofluidic flow chamber 160 includes three inlet ports 162, 164 and166, two outlet ports 168 and 170 and a central channel 172. In thisexample, a suspension including suspended particles enters from thefirst inlet port 162. Other fluid streams, such as a pair of solvent orbuffer fluid streams enter the central channel 172 from either side ofthe first inlet port 162. As shown in FIGS. 7 a-7 c, the relative flowrates of each inlet port may be modulated to vary the resulting incomingstream 174 into the central channel 172. In FIG. 7 a, for example, therelative flow rates of the streams in the second inlet port 164 and thethird inlet port 166 are relatively equal and pinch the flow from thefirst inlet port 162 at a neck and form a narrow stream of the firstfluid approximately down the center of the central channel 172. Byvarying the flow rates of the second and third inlet streams 164 and166, the width of the first fluid stream 174, i.e., the suspension, canbe narrowed down to the width of a single particle. Thus, the inletsample suspension 174 may be “prefocused” into a narrow, or even singlefile, particle stream surrounded on either side by a potentialcollection stream. This allows for a decrease in the lateral distance,i.e., distance perpendicular to the flow direction, a particle must bemoved away from the suspension stream to be captured in the collectionstream and, thus, an increase in sorting efficiency.

FIG. 7 b shows the embodiment of FIG. 7, wherein the flow rate of thethird inlet port 166 is less than the flow rate of the second inlet port164 and prefocuses the inlet particle stream in the lower half of thecentral chamber 172. Conversely, FIG. 7 b shows the embodiment of FIG.7, wherein the flow rate of the third inlet port 166 is greater than theflow rate of the second inlet port 164 and prefocuses the inlet particlestream in the upper half of the central chamber 172. The relative flowrates of the three inlets can thus be modulated to control the particlestream within the central channel.

FIG. 8 shows yet another embodiment of a microfluidic flow chamber 180in which a particle of interest may be separated from a suspension. Asin FIG. 7, the microfluidic flow chamber 180 includes three inlet ports182, 184 and 186 and a central channel 188. The chamber 180 of FIG. 8,however, includes six outlet ports 188, 190, 192, 194, 196 and 198. Thenumber of outlet ports shown in FIG. 8 is merely exemplary and mayinclude any number of outlet ports greater than or equal to two. In thisexample, the plurality of outlet ports may be used to sort a pluralityof particles into various outlet ports. Different types of particles,for example, may be sorted into different outlet ports. Alternatively,the plurality of outlet ports may be used to individually sort the sametype of particles into different outlet ports. In yet anotherembodiment, the side flows may be modulated as described above todispense particles, chemicals and/or fluids (e.g., reagents) intomultiple outlet ports for use in various downstream applications ornetworks.

Alternatively, the incoming streams may be prefocused prior to entryinto the microfluidic flow chamber, or the side inlet ports may bearranged to enter the central channel downstream of the first inletport.

FIG. 9 shows an embodiment of a microfluidic flow chamber 200 in which aparticle of interest may be separated from a suspension via a mechanicalactuator. As shown in FIG. 9, the central channel 202 includes a sidechannel 204 through which incoming fluid flow is controlled by a valve206. After a particle is detected, the valve may be opened to vary thefluid flow within the central channel 202 and divert the suspensionalong with the particle away from the first outlet port 208 into thesecond outlet port 210. Alternatively, the valve 206 may be closed orthe flow through the valve may be merely adjusted to divert the particleinto the desired outlet port. Similarly, the valve 206 may be positionedon the opposite side of the central chamber 202 and may obtain a similarresult by providing or modulating the flow in the opposite direction.

FIGS. 9 a-9 c show yet another embodiment of a microfluidic flow chamber220 in which a particle of interest may be separated from a suspensionvia a mechanical actuator. As shown in FIG. 9 a, the particle 222 entersthe central channel 224 in the suspension via the first inlet port 226.In FIG. 9 b, the valve 228 activates after the particle is identified asdescribed above and redirects the particle 222 into the second outletport 230. Then, in FIG. 9 c, after the particle 222 has exited thecentral channel 224, the valve 228 retracts and the fluid stream flowsreturn to their steady state condition.

FIGS. 9 d-9 f show an exemplary microfluidic flow chamber 240 in which aparticle of interest may be separated from a suspension via a chemicalactuator. As shown in FIGS. 9 d 9 f, the microfluidic flow chamber 240includes a chemical actuator material 242, such as a hydrogel, thatswells or shrinks in reaction to an attribute associated with aparticular particle of interest (e.g., pH). Hydrogels, such as these areknown in the art. Beebe, David J. et al, “Functional Hydrogel Structuresfor Autonomous Flow Control Inside Microfluidic Channels, Nature, vol.404, pp. 588-90, (Apr. 6, 2000), for example, discloses hydrogelactuators that may be used in the present embodiment.

FIGS. 9 d-9 f show a chemically actuated valve 244 including thechemical actuator material 242. In FIG. 9 d, for example, the chemicalactuator is in its normal condition in which the valve 244 is open andthe suspension flows through the first outlet port 246. FIG. 9 e showsthe chemical actuator in its active state in which the chemical actuatormaterial 242 is swollen in response to a detected attribute, effectivelyshutting off the first outlet port 246 and the suspension flows throughthe second outlet port 252 and allowing the particle 250 of interest tobe collected. Although FIG. 9 e shows the chemically actuated valve 244completely closing off the first outlet port, the swelling of thechemically actuated material 242 may also merely create a barrier toparticular-sized particles while allowing the remainder of thesuspension to pass into the first outlet port 246. Where the individualvalve members are angled toward the second outlet port 252, the blockedparticles 250 may be conveyed to the second outlet port 252 forcollection. FIG. 9 f further shows the chemically actuated valve 244returned to its open condition after the detected particle 250 haspassed into the second outlet port 252.

Alternatively microfluidic flow devices may employ laminar flows andspecific microgeometries for non-actuated separation of colloidal and/orcellular particles in fluid suspensions. The geometry of these deviceshas been designed to act similarly to a filter without the use ofmembranes or sieves which are highly susceptible to clogging andfouling. Such devices will also be capable of replacing thecentrifugation step common to many biological processes upon a chipsurface. With a microscale alternative to centrifugation available, ahost of multi-step biological processes such as bead-based assays andcell counting using dying techniques will be able to be performed withinmicrofluidic devices

As demonstrated in FIGS. 10 12, specific channel geometries may becreated to take advantage of the laminar nature of fluids flowing inmicrochannels. In each of these designs, the particle suspension entersthe central channel 260 through a first inlet port 262. A second fluidstream, such as a solvent stream, enters the channel 260 through asecond inlet port 264, which meets the first inlet port 262 at anyangle. Because of the laminar nature of microfluidic flows, thesestreams will generally not mix convectively. The central channel 260further includes microscale obstacles 265. Molecular debris small enoughto fit through the openings formed by the microscale obstacles 265 willbe carried down the first outlet port 266. Due to the presence ofmicroscale obstacles, however, any particles larger than the separationof the obstacles will be shuffled toward the second outlet port 268 andexit the central channel 260 with a portion of the second fluid stream.The designs shown here do not depend upon relative channel size, insteadthe presence of the microscale obstacles at or near the confluence ofthe two (or more) inlet streams alter the direction of flow for anyparticulate matter in the suspension inlet stream(s).

FIG. 13 further shows a configuration for sorting particles in thesuspension by size and produces a size fractionation effect by designingthe size of the gaps 274 between the guides 276 to increase away fromthe first inlet port 262, by which the suspension is introduced into thecentral channel 270. By gradually increasing the widths of the gaps 274moving away from the first inlet port 262, particles of increasing sizeflow into the guides 276 and may be collected individually.

FIG. 14 shows yet another embodiment of a non-actuated separation ofmotile particles within a suspension between laminar flows. In thisembodiment, motile particles 280 entering in the suspension flow 282move within the suspension flow and can pass from the suspension flow282 into the second fluid stream 284 without the need of an actuator toseparate the particles 280 from the suspension flow 282. In this manner,the motile particles 280 may enter the second fluid stream 284 and exitthe central channel 286 through the second outlet port 290 instead ofthe first inlet port 288. For example, in a suspension 282 containingsperm, the active sperm may move on their own into the second fluidstream 284 for collection, while inactive sperm are carried out of thecentral channel 286 with the suspension 282 via the first outlet port288.

Non-actuated separation of colloidal and/or cellular particles from asuspension in a microfluidic flow device presents a very simple approachto microfluidic separations or enrichments of colloidal and/or cellularparticles because it relies upon the condition native to fluids flowingon the microscale, regardless of flow rate or channel morphology:laminar flows. Furthermore, the selection of materials for theconstruction of these devices is irrelevant, thus they may beincorporated into microfluidic devices constructed on any substrate.

FIG. 15 shows another example of a microfluidic flow chamber in which aseries of discrete sample suspensions 300 are combined into a singlelaminar flow. In this example, a plurality of discrete samples 300 formthe single sample flow. The sample flow further preferably includesbuffers 302 between each discrete sample 300 to preventcross-contamination between samples 300. In this manner, a singlemicrofluidic flow chamber 304 can separate particles from a series ofsamples to increase throughput. The series of discrete samplesuspensions may, for example, be created using a microfluidic dispenseras shown and described above with reference to FIG. 8 in whichindividual samples are directed into a plurality of outlet ports andcombined downstream into a series of discrete sample streams.

FIG. 16 shows a cartridge 310 that may be plugged into, or otherwiseconnected to, a system for separating one or more colloidal or cellularparticles from a suspension. The cartridge 310 may be reusable ordisposable. The cartridge may include a sample reservoir 312, or otherinlet mechanism, for receiving a fluid suspension. The sample reservoir312 is connected to a central channel 314 via a first inlet port 316.The cartridge further includes a waste receptacle 318, or other outletmechanism, connected to the central channel 314 via a first outlet port320 for receiving the suspension after it has passed through the centralchannel 314 for the removal of one or more particles of interest. Acollection receptacle 322 is also connected to the central channel 314via a second outlet port 324 for receiving the particles collected fromthe suspension. The collection receptacle 322 may include a reservoir orother means for holding the collected particles or may include a channelor other means for providing the collected particles to downstreamnetworks for further processing.

The cartridge 310 may also include a second inlet reservoir 326 forreceiving a second fluid, may receive the second fluid from an externalsource in the system, or may not utilize a second fluid at all, such asdescribed with reference to FIG. 5. If used, the second fluid mayinclude a fluid such as a buffer or a solvent (e.g., water, a salinesuspension and the like) or a reagent (e.g., antibody tagged particles,fluorescent tags, lysing agents, anticoagulants and the like), or anycombination thereof. Indeed, the fluid requirements may besystem-specific and may be matched to the intended application and modeof use. The second inlet reservoir 326 or receptacle for receiving asecond fluid, if used, may be connected to the central channel 314 via asecond inlet port 328.

The reservoirs or receptacles may include any interface for transferringa fluid known in the art. For example, the reservoir may be adapted toreceive fluids from a syringe, either with or without a needle, from atube, from a pump, directly from a human or animal, such as through afinger stick, or from any specially designed or standard fluid transfercoupling.

The microfluidic flow chambers described herein may be manufactured by avariety of common microelectronics processing techniques. A pattern of ashadow mask may be transferred to a positive or negative photoresistfilm spun upon a silicon wafer, a glass slide, or some other substrate,for example. This pattern may be sealed and used directly as themicrofluidic network, replicated in another material, or furtherprocessed. The substrate may be further processed through subsequent wetetching, dry etching, molecular epitaxy, physical deposition ofmaterials, chemical deposition of materials, and the like, or anycombination of these or similar techniques. The final network may beused directly or reproduced through the use of a replication techniquedesigned to produce a replica upon the master, such as by the pouringand curing, imprinting in or deposition of elastomers, polymers and thelike. A pump or other means for introducing and controlling fluid flowwithin the fluidic network as well as a means for connecting the pump orpressure differential means may also be provided. The network canfurther be sealed, such as with a cover slip, glass slide, siliconwafer, polymer films or a similar substrate.

In one specific, nonlimiting example, a pattern on a shadow mask wasexposed to ultraviolet light and transferred to a negative photoresistfilm spun upon a silicon wafer to a depth of approximately 5 μm. Atwo-part mixture of poly(dimethyl siloxane) (PDMS), which iscommercially available from Dow Corning under the trade name of Sylgard184, was poured and cured upon the silicon master to produce a flexible,biocompatible optically transparent replica. In addition to the PDMSchannel network a flow apparatus comprising a syringe pump such as akdScientific, model 200 syringe pump and a polymethyl methalacrylate(PMMA) flow introduction base. The PDMS channel network was placed uponthe PMMA base, and holes were punched through the PDMS to provide accessfor the microchannels to the ports in the base. The network was furthersealed with a cover slip. Because the PDMS forms a tight seal with bothPMMA and glass, no additional bonding or clamping was required. Thesyringe pump was further fitted with 3 cm³ plastic syringes (such asavailable from Becton-Dickson) joined to the base.

One embodiment of an optical trap and digital microscopy that may beused with the microfluidic flow devices described herein may incorporatea piezoelectric mirror (such as available from Physik Instrumente, modelS-315) to simultaneously trap several particles by rapidly scanning asingle laser beam (such as available from Spectra Physics, 532 nm,typically operated at 200 mW) among a number of positions to create atime-averaged extended trapping pattern. A Neofluar, 100×, oil immersionhigh numerical aperture objective (N.A.=1.30) can be used to focus thebeam and create the optical trap. CCD images can be captured by a dataacquisition board and processed by LabView (National Instruments)routines that may be customized to distinguish various visual particleor cell features for specific applications. Optical traps and digitalmicroscopy are described in further detail, for example, in Mio, C.;Gong, T.; Terry, A.; Marr, D. W. M., Design of a Scanning Laser OpticalTrap for Multiparticle Manipulation, Rev. Sci. lnstrum. 2000, 71,2196-2200.

While the invention has been particularly shown and described withreference to particular embodiment(s) thereof, it will be understood bythose skilled in the art that various other changes in the form anddetails may be made without departing from the spirit and scope of theinvention. One skilled in the art of microfluidic flows, for example,would recognize that downstream or upstream analogues of mechanismsdescribed herein may be substituted for the particular exemplarystructures disclosed herein.

1. A flow-through device for sorting cells in a fluid sample comprising:an inlet for receiving said fluid sample, said inlet being coupled to achannel comprising a plurality of obstacles arranged in rows, whereineach subsequent row of obstacles is shifted laterally with respect to aprevious row, wherein the obstacles are adapted to differentially directa first cell type from said fluid sample to a first direction and asecond cell type from said sample to a second direction, wherein saidchannel comprises a first outlet in said first direction and a secondoutlet in said second direction.
 2. The device of claim 1, wherein saidrows comprise an ordered array of obstacles, whereby said array isasymmetric with respect to the average direction of a field, such thatcells having a size less than a predetermined critical size aretransported in a first direction, and cells having a size at least thatof the critical size are transported in a second direction, wherein thefirst and second directions are different, thereby separating said cellsaccording to size.
 3. The device of claims 1, or 2, wherein said deviceis configured to provide a modulated flow rate.
 4. The device of claim3, wherein said flow rate is achieved by electrical, electro-osmotic,gravitational, hydrodynamic, pressure gradient, or capillary action. 5.The device of claim 2, wherein said ordered array of obstacles is tiltedat an offset angle with respect to direction of said flow-through.
 6. Amethod for manufacturing a flow-through device for cell sortingcomprising: providing a substrate; creating a pattern of obstacles onsaid substrate, wherein said obstacles are arranged in rows and whereineach subsequent row of obstacles is shifted laterally with respect to aprevious row, wherein said obstacles allow passage of cells based ontheir size, shape or deformability; and creating at least two outletswherein said first outlet is adapted for output of larger cells and saidsecond outlet is adapted for output of said smaller cells.
 7. The methodof claim 6, wherein said device is configured to allow a field whichprovides a modulated flow rate through said flow-through device.
 8. Themethod of claim 7, wherein said field is electrical, electro-osmotic,gravitational, hydrodynamic, pressure gradient, or capillary action. 9.The method of 8, wherein said field is an electrical field.
 10. Themethod of claim 9, wherein said rows comprise an ordered array ofobstacles, whereby said array is asymmetric with respect to the averagedirection of a field, such that cells having a size less than apredetermined critical size are transported in a first direction, andcells having a size at least that of the critical size are transportedin a second direction, wherein the first and second directions aredifferent, thereby separating said cells according to size.